PROJECT SUMMARY We are testing a novel strategy to regenerate damaged descending bulbospinal respiratory axons and reinnervate phrenic motor neurons (PhMN) after cervical spinal cord injury (SCI) in rats. SCI is caused by trauma to the spinal cord, and more than half of all cases occur in the cervical region, leading to breathing compromise by damaging circuits involved in respiratory control. Restoration of functional deficits caused by SCI is limited due to the low intrinsic drive of neurons to regenerate axons and a lack of guidance cues to signal growing axons to appropriate targets, among others. The C3-C5 mid-cervical spinal cord levels house the PhMNs, which are responsible for diaphragm activation. PhMNs are predominately mono-synaptically innervated by supraspinal respiratory neurons located in a brainstem nucleus called the rostral Ventral Respiratory Group (rVRG). We are seeking to reverse respiratory dysfunction after SCI by restoring the crucial circuit controlling PhMNs, and thus diaphragm activation. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family of growth factors, promotes axonal growth and acts as a guidance cue. Phosphatase and tensin homolog (PTEN) is a negative regulator of mammalian target of rapamycin (mTOR), which is responsible for pro-growth pathways including axon growth. Downregulation of PTEN has been shown to induce axon regeneration. We aim to promote targeted reinnervation of PhMNs and restore diaphragm function by systemically inhibiting PTEN with antagonist peptides to induce axon growth through the injury, followed by BDNF overexpression selectively in PhMNs via an adeno-associated virus (AAV) to direct growing axons. In Aim 1, we will determine whether providing a PhMN-specific source of the axon guidance molecule, BDNF, promotes targeted PhMN reinnervation by rVRG axons following cervical SCI. We will assess rVRG axons using an AAV vector expressing a dual anterograde/trans-synaptic tracer, examining regrowth and collateral sprouting, and identify synaptic reconnection with spared PhMNs by post-synaptic accumulation of the trans-synaptic marker. In Aim 2, we will determine whether rVRG-PhMN circuit re-connectivity promotes diaphragmatic recovery after cervical SCI. We will assess the ability of rVRG-PhMN reconnection to restore diaphragm function by testing in vivo diaphragm activation via electromyography (EMG). Excitingly, we can distinguish between modes of recovery, i.e. ipsilateral regrowth versus contralateral sprouting, by selectively silencing unilateral rVRG neurons with inhibitory Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) and recording any subsequent changes in EMGs. BDNF overexpression is associated with neuropathic pain and abnormal motor function. We will test for unintended consequences of BDNF, including pain phenotypes and motor gains/deficits. We aim to use the strategy proposed here to reconnect motor neurons responsible for diaphragm activation with respiratory centers in the medulla to restore respiratory function following disruption after cervical SCI. The potential therapeutic benefits being explored will have profound implications for SCI patients suffering respiratory dysfunction.